This is a proposal to investigate assembly of fibronectin (FN), a key informational protein of extracellular . matrix. The first specific aim is to characterize binding of the N-terminal modules of FN to labile molecules on cell surfaces where initial FN assembly occurs. The cell surface molecules have an extremely Large Apparent Molecular Mass when analyzed as a complex with sodium dodecyl sulfate (SDS) and are called LAMMs. The second aim is to learn the roles of integrins in control of FN assembly. The research plan is based on four recent findings. First, we have characterized a 49-residue peptide, FUD, as a potent inhibitor of FN assembly. FUD is derived from MSCRAMM (Microbial Surface Component Recognizing Adhesive Matrix Molecules) Fl of Streptococcus pyogenes. FUD binds to N-terminal type I modules of FN that are critical for assembly of FN. Analyses of the FUD-FN interaction will test the hypothesis that extensible segments of MSCRAMM F1 and LAMMs interact similarly with FN. Second, we have generated FN-null fibroblasts in which to re-express various splice variants of FN as a fusion with green fluorescent protein (GFP) and thereby learn the requirements for assembly of endogenous FN and the role of endogenous FN in the assembly of exogenous FN and trafficking of integrins. Third, we discovered that appropriately stimulated adherent human platelets assemble FN by a mechanism that involves LAMMs. Platelets can be obtained in quantity, are in essence packets of contractile and adhesive machinery, and will be the primary source from which to purify and characterize LAMMs using the tools of proteomics and genomics. Fourth, we have developed a series of cell lines that express mutant b1A integrin subunit with altered phosphorylation potential and function. These cell lines will make it possible to dissect relationships among roles of b1A integrins in cell adhesion, formation of focal and ECM contacts, FN assembly, and cell migration.